Measuring toxicant effects on cellular function in a microarray format

Project Summary/Abstract – The EPA maintains a Toxics Release Inventory of andgt;650 toxic chemicals that are disposed or released from andgt;20,000 industrial sites in the United States, while HHS and USDA maintain a list of over 60 select agents and toxicants that pose a severe risk to human, animal, and plant health. These compounds represent only a fraction of the known and unknown environmental toxicants that may adversely affect human health. New and uncharacterized toxicants are synthesized at an unprecedented rate, and trace quantities of these are rapidly entering the environment. There is thus a critical need for a high-content assay of the effects of toxicants on cell function. Such a platform would broaden our understanding of environmental components that may represent risks for autoimmune disease, immunodeficiencies, and neoplasia. Stem cells are an important and sensitive target for toxicants as they are critical components of embryonic development and are integral to the maintenance of adult tissues. Current assays measuring changes in stem cell differentiation and programming in response to toxic effects provide insight into the pathogenesis of toxicant exposure, but these techniques are not capable of the throughput necessary to stay apace the speed of new toxicants entering the environment. Further, the expense of performing the labor-intensive gold- standard tests limits the amount of data gathered for a cell population, toxicant identity, and exposure concentration. Nonetheless, these tests are regularly performed since such measurements are critical to our understanding of the risks these agents represent, and to our ability to moderate or eliminate those risks. The goal of this project is the development of instrumentation capable of detecting toxicant effects on stem cell differentiation and behavior in a sensitive high-content assay. This assay will be based on the detection of changes in the surface marker expression and cytokine excretion profile of a spatially encoded microarray of stem cells. These cells will be exposed to one or more simultaneous toxicant concentration gradients, which will represent the spectrum of exposures or two-agent co-exposures that might be encountered by cells in the environment. Ciencia is proposing instrumentation that can measure both grating-coupled surface plasmon resonance as well as plasmonically-enhanced fluorescent emission from three distinct fluorophores. This system will also incorporate a zoom lens that permits whole-chip imaging and high-magnification single-spot images. It will support on-chip incubation and a sample chamber that permits exposure of cells to a diffusing gradient of toxicant, making it ideal for the laboratory evaluation of toxicant effects on stem cell differentiation. Concurrent assessments of effects on other cell lineages may also performed. This proposed instrumentation would be highly versatile, with modular components suggesting additional utility as the project progresses to Phase III. A well-validated platform of this nature may be readily applicable to drug discovery efforts and lead compound validation, and may ultimately find value as a clinical diagnostic.Project Narrative: There is a critical need for highly sensitive and specific techniques that are capable of defining the risk levels to human health that result from exposure to a broad range of toxicants found in the environment. To address this need, we propose to build upon our extensive experience with microarray Surface Plasmon Resonance (SPR) systems to develop a high-content instrument platform that can assess the impact of toxicant exposure on stem cell differentiation that is not constrained by the limitations of existing technologies. This multimode, multi-fluor SPR-based analytic instrument will use small volumes of sample and reagents to identify and quantitate the effects of toxicant exposure on stem cell functioning without foreknowledge of the contaminant’s identity or toxic concentration profile. This technology will thus be suitable for use in both research and clinical laboratories, and with further development will be extensible to a point-of- care environment.